This invention is related to germacrene C synthase genes and related compositions and methods.
Volatile metabolites of Lycopersicon species are of interest because of their roles in tomato flavor and in host defense, for example against plant pathogens, e.g., microbial pathogens, and pests, e.g., arthropod and mammalian herbivores (Buttery et al., ACS Symp. Ser. 52:22-34, 1993; Buttery et al., J. Agric. Food Chem. 38:2050-2053, 1990; Carter et al., J. Agric. Food Chem. 37:1425-1428, 1989; Lin et al., J. Chem. Ecol. 13:837-850, 1987; and Carter et al., J. Agric. Food Chem. 37:206-210, 1989). Whereas few volatile terpenoids are found in fruit (Buttery et al., ACS Symp. Ser. 52:22-34, 1993; and Buttery et al., J. Agric. Food Chem. 38:2050-2053, 1990), the leaf glandular trichomes produce a rich spectrum of monoterpenes and sesquiterpenes. Nearly twenty monoterpenes, including limonene, have been found in the leaves of the domestic tomato L. esculentum (Buttery et al., J. Agric. Food Chem. 35:1039-1042, 1987; and Lundgren et al., Nord. J. Bot. 5:315-320, 1985), and most are also present in wild tomato species (Lundgren et al., Nord. J. Bot. 5:315-320, 1985). A number of C13 norsesquiterpenoid glycosidic ethers have been reported in tomato fruit (Marlatt et al., J. Agric. Food Chem. 40:249-252, 1992); these compounds are derived by degradation of carotenoids (Isoe et al., Helv. Chim. Acta 56:1514-1516, 1973). The sesquiterpene content of tomato leaf oil varies considerably among species, with caryophyllene and humulene being widespread and reported from L. esculentum, L. hirsutum, L. pimpinellifolium, L. peruvianum, L. cheesmanii, L. chilense and L. chumielewski (Lundgren et al., Nord. J. Bot. 5:315-320, 1985). The epoxides of both of these sesquiterpenes are also present in L. esculentum, as is a low level of xcex4-elemene (Buttery et al., J. Agric. Food Chem. 35:1039-1042, 1987). Various accessions of L. hirsutum contain xcex1-copaene, xcex3-elemene, zingiberene and xcex1-santalene as major leaf oil sesquiterpenes (Lundgren et al., Nord. J. Bot. 5:315-320, 1985). Germacrenes have not been reported in the genus Lycopersicon.
The terpenoid composition of the highly disease resistant L. esculentum cv. xe2x80x98VFNT Cherryxe2x80x99 (a tomato of multi-species pedigree carrying resistance to Verticillium dahliae, Fusarium oxysporum, root-knot Nematode, Tobacco mosaic virus and Alternaria stem canker) has not been examined, although it is an important breeding line (Jones et al., HortSci. 15:98, 1980).
A germacrene C synthase gene from Lycopersicon esculentum has been cloned and sequenced. Transgenic expression of this gene in cells of a plant results in, for example, increased resistance to pathogens. Such pathogens include, for example, viruses, bacteria, spots, wilts, rusts, mildews, and related fungi as well as nematodes. Transgenic expression also will alter flavor and odor profile, which will deter eating of the plants by herbivores, such as Coleopteran, Lepidopteran, Homopteran, Heteropteran, and Dipteran pests, as well as acarine plant mites and related arthropods, mollusks, and mammalian herbivores. Additionally, expression of germacrene C synthase may increase the neutraceutical value of the plant and may also enhance the plant""s attractiveness for pollinators, and possible for insect predators.
The invention includes a purified protein having a primary amino acid sequence as shown in FIG. 4 (SEQ ID NO:2). This protein displays germacrene C synthase biological activity as described herein.
Also encompassed within the invention are proteins with a primary amino acid sequence that differs from the sequence as shown in FIG. 4 (SEQ ID NO:2) only by one or more conservative amino acid substitutions.
Also included are polypeptide fragments that comprise less than the full length of the protein as shown in FIG. 4 (SEQ ID NO:2). Such fragments, may be, for example, at least 10, at least 15, at least 20 at least 30, at least 50, or at least 75 amino acids in length. Such polypeptide fragments may be used for example, as immunogens to raise antibodies that may be used in research and diagnostic applications.
The invention also includes proteins and polypeptide fragments that show specific degrees of sequence similarity with the sequence as shown in FIG. 4 (SEQ ID NO:2) and that have germacrene C synthase biological activity. Such similarity may be, for example, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% similarity as measured by standard analysis software (e.g., Blastp, as discussed herein, using default parameters).
Also included are isolated nucleic acids that encode the above proteins and fragments thereof. Such nucleic acids include a nucleic acid with the sequence as shown in FIG. 6 (SEQ ID NO:1) and degenerate versions of such a nucleic acid. Such nucleic acids may encode a polypeptide with germacrene C synthase biological activity.
Also encompassed within the invention are polynucleotides of at least 15 nucleotides in length (e.g., at least 17, at least 30, at least 50, or at least 100 nucleotides in length) of the sequence as shown in FIG. 6 (SEQ ID NO:1). Such polynucleotides may be used, for instance, as probes or primers as described herein. Probes may be labeled with, for instance, radioactive, fluorescent, or biotin-avidin labels and used for the detection of nucleic acids that show a certain degree of similarity to the probe sequence, thereby detecting nucleic acid sequences related to the germacrene C synthase gene of the invention.
The invention also includes nucleic acids and polynucleotide fragments (of at least 15, at least 17, at least 30, at least 50, or at least 100 nucleotides in length) that hybridize under defined conditions of stringency with the nucleic acid sequence as shown in FIG. 6 (SEQ ID NO:1). Such conditions for stringency may include, for example, prehybridization at 65xc2x0 C. for 4 h, followed by hybridization at 65xc2x0 C. overnight, followed by washing at 65xc2x0 C. in 2xc3x97NaCl-NaH2PO4-EDTA buffer with 0.5% SDS for 20 min. Nucleic acids of the invention may also hybridize with the sequence as shown in FIG. 6 (SEQ ID NO:1) under less stringent wash conditions, for instance conditions of 60xc2x0 C. and 0.5xc3x97SSC; 55xc2x0 C. and 0.5xc3x97SSC; 50xc2x0 C. and 2xc3x97SSC or even 45xc2x0 C. to as low as room temperature and 2xc3x97SSC.
The invention also encompasses recombinant nucleic acids that include a nucleic acid (or polynucleotide) as described above. Such a recombinant nucleic acid may include a promoter sequence operably linked to a nucleic acid (or polynucleotide) of the invention such that a protein is expressed under appropriate conditions.
The invention also includes cells and organisms (e.g., plants) that contain such recombinant nucleic acids. Such plants may display enhanced pathogen (and/or herbivore) resistance and may also show altered flavor or odor.
According to another embodiment of the invention, methods are provided for expressing a germacrene C synthase polypeptide in a cell. Such methods including the steps of providing a cell that comprises a polynucleotide that includes a polypeptide-encoding sequence that encodes a polypeptide with germacrene C synthase biological activity and that has at least 70% amino acid sequence identity with a native germacrene C synthase polypeptide or a homolog thereof; and culturing the cell under conditions suitable for expression of the polypeptide.
According to another embodiment of the invention, methods are provided for producing a plant having an altered phenotype selected from the group consisting of altered flavor, altered odor, and increased defense against a pathogen or herbivore. The method comprises providing a plant comprising a polynucleotide as described above and growing the plant under conditions that cause expression of the polypeptide.
According to another embodiment of the invention, methods are provided for obtaining a germacrene C synthase gene or an allele or homolog thereof. Such methods comprise the steps of contacting a polynucleotide of an organism under stringent hybridization conditions with a probe or primer comprising a polynucleotide that includes at least 15 consecutive nucleotides of a germacrene C synthase gene of FIG. 6 (SEQ ID NO:1) that hybridizes specifically to the germacrene C synthase gene of FIG. 6 (SEQ ID NO:1) or an allele or homolog thereof. This causes the probe or primer to hybridize to a gene of the organism. The methods also include isolating the gene of the organism to which the probe or primer hybridizes.
The foregoing and other objects and advantages of the invention will become more apparent from the following detailed description and accompanying drawings.
SEQ ID NO:1 shows the nucleotide sequence of the cDNA insert of pLE20.3 (see FIG. 6).
SEQ ID NO:2 shows the amino acid sequence of the germacrene C synthase protein corresponding to the open reading frame of pLE20.3 (see FIGS. 4 and 7).
SEQ ID NO:3 shows the nucleotide sequence of the cDNA insert of pLE14.2 (see FIG. 8).
SEQ ID NO:4 shows the amino acid sequence corresponding to the open reading frame of the cDNA insert of pLE14.2 (see FIG. 9).
SEQ ID NO:5 shows the degenerate forward primer used to amplify xe2x80x98VFNT Cherryxe2x80x99 leaf library cDNA by PCR as described herein (xe2x80x9cPCR-Based Probe Generation and cDNA Library Screening.xe2x80x9d)
SEQ ID NO:6 shows the degenerate reverse primer used to amplify xe2x80x98VFNT Cherryxe2x80x99 leaf library cDNA by PCR as described herein (xe2x80x9cPCR-Based Probe Generation and cDNA Library Screening.xe2x80x9d)